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HIGH PERFORMANCE THIN

LAYER

CHROMATOGARPHY(HPTLC)

1 Dr. A.R.Bekhradnia

HIGH

PERFORMANCE THIN LAYER

CHROMATOGARPHY

(HPTLC)


THIN LAYER CHROMATOGRAPHY (TLC)


Dr. A.R.Bekhradnia 4

Chromatography is a physical process of

separation in which the components to be

separated are distributed between 2 immiscible

phases a stationary phase which has a large

surface area and mobile phase which is in

constant motion through the stationary phase.

THIN LAYER CHROMATOGRAPHY (TLC)


• Mikhail Tsvet

• Born 14 May 1872

• Asti, Italy

• Died 26 June 1919 (age 47)

• Nationality Russia

• Fields botany

• Mikhail Semyonovich Tsvet (Михаи́л Семёнович Цвет, also spelled Tsvett, Tswett, Tswet, Zwet, and Cvet) (1872–1919) was a Russian-Italian botanist who invented adsorption chromatography. 6 Dr. A.R.Bekhradnia

LAAQ-B-LC001B 7

Invention of Chromatography by M. Tswett

Ether

CaCO3

Chlorophyll

Chromatography

Colors

Dr. A.R.Bekhradnia

LAAQ-B-LC001B 8

Comparing Chromatography to the Flow of a River...

Base

Water flow Light leaf

Heavy stone

Dr. A.R.Bekhradnia

LAAQ-B-LC001B 9

Chromato-graphy / -graph / -gram / -grapher

Chromatography: Analytical technique Chromatograph: Instrument Chromatogram: Obtained “picture” Chromatographer: Person

Dr. A.R.Bekhradnia

LAAQ-B-LC001B 10

Three States of Matter and Chromatography Types

Mobile phase

Gas Liquid Solid

Stationary phase

Gas

Liquid

Solid

Gas chromatography

Liquid chromatography

Dr. A.R.Bekhradnia

LAAQ-B-LC001B 11

Liquid Chromatography

Chromatography in which the mobile phase is a liquid. The liquid used as the mobile phase is

called the “eluent”. The stationary phase is usually a solid or a

liquid. In general, it is possible to analyze any

substance that can be stably dissolved in the mobile phase.

Dr. A.R.Bekhradnia

LAAQ-B-LC001B 12

Interaction Between Solutes, Stationary Phase, and Mobile Phase

Differences in the interactions between the solutes and stationary and mobile phases enable separation.

Solute

Stationary phase Mobile phase

Degree of adsorption, solubility, ionicity, etc.

Dr. A.R.Bekhradnia

LAAQ-B-LC001B

Classification

According to the force of separation: Adsorption chromatography Partition chromatography Ion exchange chromatography Gel filtration chromatography Affinity chromatography


LAAQ-B-LC001B 14

Column Chromatography and Planar Chromatography

Separation column

Packing material

Column Chromatography

Paper or a substrate coated

with particles

Paper Chromatography Thin Layer Chromatography (TLC)

Dr. A.R.Bekhradnia

15

O

utpu

t co

ncen

tratio

n

Time

Chromatogram

Dr. A.R.Bekhradnia

16

tR

t0

Inte

nsity

of d

etec

tor s

igna

l

Time

Peak tR : Retention time

h

A

t0 : Non-retention time

A : Peak area h : Peak height

Dr. A.R.Bekhradnia

LAAQ-B-LC001B 17

Separation Process and Chromatogram for Column Chromatography

Out

put

conc

entra

tion

Time

Chromatogram

Dr. A.R.Bekhradnia

Once the solvent is within ~1-2 cm of the top of

the TLC sheet, the TLC is removed from the

developing chamber and the farthest extent of

the solvent (the solvent front) is marked with a

pencil.

The solvent is allowed to evaporate from the

TLC sheet in the hood.

The spots are visualized using a UV lamp.

A fluorescent compound, usually Manganese-

activated Zinc Silicate, is added to the adsorbent

that allows the visualization of spots under a

blacklight (UV254). The adsorbent layer will

fluoresce light green by itself, but spots of analyte

quench this fluorescence and appear as a dark spot.

THIN LAYER CHROMATOGRAPHY

http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html


THIN LAYER CHROMATOGRAPHY - Visualization As the chemicals being separated may be

colorless, several methods exist to visualize the spots:

• Visualization of spots under a UV254 lamp. The

adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.

• Iodine vapors are a general unspecific color.

• Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate.

• Once visible, the Rf value of each spot can be determined

Chromatogram of 10 essential oils, Stained with vanillin reagent.


http://en.wikipedia.org/wiki/Image:TLC-Essential-Oils.jpg

THIN LAYER CHROMATOGRAPHY Calculation of Rf’s

The Rf is defined as the distance the center of the spot moved divided

by the distance the solvent front moved (both measured from the origin)

A B CU

x xx x

Solvent Front

Origen

Distance solvent

migrated = 5.0 cm

Distance A

migrated = 3.0 cm

Distance B

migrated = 2.0 cm

Distance C

migrated = 0.8 cm0.8 cm

3.0 cm

Rf (A) =

Rf (B) =

Rf (C) =

Rf (U1) =

Rf (U2) =

2.0 cm

5.0 cm= 0.40

= 0.60

= 0.16

= 0.60

= 0.16

3.0 cm

5.0 cm

0.8 cm

5.0 cm

3.0 cm

5.0 cm

0.8 cm

5.0 cm

D

x

Rf (D) = = 0.804.0 cm

5.0 cm

4.0 cm


Rf values can be used to aid in the identification of a

substance by comparison to standards.

The Rf value is not a physical constant, and

comparison should be made only between spots on

the same sheet, run at the same time.

Two substances that have the same Rf value may be

identical; those with different Rf values are not

identical.

THIN LAYER CHROMATOGRAPHY – Rf’s


Absorption of Solutes

The adsorption strength of compounds increases with increasing polarity of functional groups, as shown below: -CH=CH2, -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONR2, -CO2H.

(weakly adsorbed) (strongly adsorbed)

(nonpolar) (more polar)

THIN LAYER CHROMATOGRAPHY – Rf’s

Elution Strength of Mobile Phase (ε)

Elution strength is generally considered to be equivalent to polarity. A solvents elution strength depends on Intermolecular Forces between the solvent and the analytes and between the solvent and the stationary phase.

A more polar (or more strongly eluting solvent) will move all of the analytes to a greater extent, than a less polar, weakly elution solvent.

For example, the elution strength of hexane is very low; ε = 0.01. the elution strength of ethyl acetate is higher; ε = 0.45 the elution strength of ethanol is even higher; ε = 0.68


Solvent MF MW

Bp (oC)

Density (g/mL) Hazards* Dipole Elution

Stength

(ε)

Hexane CH3(CH2)4CH3

C6H14 86.17

68.7 0.659

Flammable Toxic

0.08 0.01

Toluene C6H5CH3

C7H8 92.13

110.6 0.867

Flammable Toxic

0.31 0.22

Diethyl ether CH3CH2OCH2CH3

C4H10O 74.12

34.6 0.713

Flammable Toxic, CNS Depressant

1.15 0.29

Dichloromethane CH2Cl2

CH2Cl2 84.94

39.8 1.326

Toxic, Irritant Cancer suspect

1.14 0.32

Ethyl Acetate CH3CO2CH2CH3

C4H8O2 88.10

77.1 0.901

Flammable Irritant

1.88 0.45

Acetone CH3COCH3

C3H6O 58.08

56.3 0.790

Flammable Irritant

2.69 0.43

Butanone CH3CH2COCH3

C4H8O 72.10

80.1 0.805

Flammable Irritant

2.76 0.39

1-Butanol CH3CH2CH2CH2OH

C4H10O 74.12

117.7 0.810

Flammable Irritant

1.75 0.47

Propanol CH3CH2CH2OH

C3H8O 60.09

82.3 0.785

Flammable Irritant

1.66 0.63

Ethanol CH3CH2OH

C2H6O 46.07

78.5 0.789

Flammable Irritant

1.70 0.68

Methanol CH3OH

CH4O 32.04

64.7 0.791

Flammable Toxic

1.7 0.73

Water HOH

H2O 18.02

100.0 0.998

1.87 >1

Solvent Properties and Elution Strengths


Elution Strength of Mixed Solvents

The elution strength of the mixture is assumed to be the weighted average of the elution strengths of the components: εo

net = εoA (mole % A) + εo

B (mole % B) where: mole % A = (moles A) / (moles A + moles B) Thus, to determine the εo

net of a solvent mixture, the molar ratio of the solvents must first be calculated. For example, the εo

net of a solvent mixture prepared from 1.0 mL of ethyl acetate plus 9.0 mL of hexanes is calculated as shown below: εo

net = εoEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] + εohexane [(moles hexane)/(moles EtOAc+moles hexane)] where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc] thus: εo

net = {0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)(0.659g/mL)/86.18g/mole)]} {(1.0 mLEtOAc)(0.902g/mL)/88.11g/mole) + (9.0 mLhexane)(0.659g/mL)/86.18g/mole)} and εo

net = 0.067


Resolution The separation between two analytes on a

chromatogram can be expressed as the resolution, Rs and can be determined using the following equation:

Rs = (distance between center of spots)

(average diameter of spots) In TLC, if the Rs value is greater than 1.0, the

analytes are considered to be resolved.

x x


Improving Resolution:

For two closely migrating components, optimum resolutions are usually obtained when the Rf’s of both compounds are between 0.2 and 0.5

* To Improve Rs, change the elution strength of the solvent

to optimize Rf’s • change εonet, all compounds will be effected similarly. • Alter the composition of the solvent system so that the

components affinity for the mobile phase vs. the solid phase are differentially changed (= change in selectivity). • Changing the chemical nature of the solvent system,

such as changing a hydrogen bonding solvent to a solvent which cannot hydrogen bond to the analyte, is often the most effective.

** Improve Rs by decreasing the diameter of the

analyte spots. This can be achieved by applying smaller and less concentrated spots. http://orgchem.colorado.edu/hndbksupport/

TLC/TLCprocedure.html


http://orgchem.colorado.edu/hndbksupport/

HIGH

PERFORMANCE THIN LAYER

CHROMATOGARPHY

(HPTLC)



Introduction of HPTLC

• HPTLC is the improved method of TLC which utilizes the

conventional technique of TLC in more optimized way.

• HPTLC takes place in highspeed capillary flow range of the mobile phase.

• There are three main steps HPTLC procedure, they are

1] Sample preparation, volume precision and exact position are achieved by use of suitable instrument.

2] Solvent (mobile phase) migrates the planned distance in layer (stationary phase) by capillary action. In this process sample separated into it’s components.

3] Separation tracks are scanned in densitometer with light

beams in visible or uv region


Steps Involving in HPTLC

Sample Preparation Selection of

chromatography layer

Pre-washing

Pre-conditioning

Application of sample

Chromatography development

Detection of spots

Scanning & documentation


Sample preparation

• Normal phase chromatography: non polar solvent

• Reversed phase chromatography: polar solvent

Selection of chromatography layer

Depends on nature of material to be separated Commonly used(silica gel, alumina)


Pre-washing

• It is purification step

• Mainly methanol is used

• Essential for quantitative evaluation

Linomat lV applicator



Selection of HPTLC plates

• Previously hand made plates is used in TLC for both

qualitative and quantitative work. Certain drawbacks with

that is nonuniform layer, formation of thick layer, paved for

advent of precoated plates.

• Nowadays precoated plates are available in different format

and thickness by various manufactures. Precaoted plates can

be used for both qualitative and quantitative work in

HPTLC, they are

• GLASS PLATES

• POLY ESTER/POLYETHYLYNE

• ALUMINIUM PLATES


Glass Plates: Offers superior flat and smooth surface.

- fragile

- high weight

- higher production cost

Polyester/polyethylene plates:

Thickness of plate is 0.2mm.

- It can be produced in roll forms.

- Unbreakable.

- Less packing material is required.

- Development of plate cann’t be above temperature

1200 c loses its shape.

Aluminium plates: Thickness of plate is 0.1mm.

- It can be produced in roll forms.

- Unbreakable.

- Less packaging material is required.


SORBENTS USED IN HPTLC PLATES:

sorbents which are used in convential TLC are also used in HPTLC with or without modification.

- silica gel 65F

- highly purified silicagel 60

- aluminium oxide

- cellulose microcrystalline

- silica gel

- reversed stationary phase


The layer thickness in HPTLC is around 100-

200cm,in conventional it is 250mm.

Layer prewashing: Ascending method

- Dipping method

- Continuous method


The plates are activated by placing in an oven at 1101200 C for 30 min, this step will removes water that has been physically absorbed on surface at solvent layer.

Freshly opened box of HPTLC plates usually does not require activation.

Activation at higher temp and for longer time is avoided which leads to very active layer and there is risk of sample being decomposed


- Methanol (commonly used)

- Chloroform:methanol:ammonia(90:10:1)

- Chloroform:methanol(1:1)

- Methylene chloride:methanol(1:1)

- Ammonia(1%)solution


Usual concentration range is 0.1-1µg / µl,above

this causes poor separation.

Linomat IV (automatic applicator) - nitrogen gas

sprays sample and standard from syringe on TLC

plates as bands.

Band wise application - better separation - high

response to densitometer.


Processes in the Developing Chamber The «classical» way of developing a chromatogram is to place the plate in a chamber, which contains a sufficient amount of developing solvent. The lower end of the plate should be immersed several millimeters. Driven by capillary action the developing solvent moves up the layer until the desired running distance is reached and chromatography is stopped. The following considerations primarily concern silica gel as stationary phase and developments, which can be described as adsorption chromatography.


http://www.camag.com/media/4207P5N3/entwicklungskammer_vorgang.png

Provided the chamber is closed, four partially competing processes occur: Between the components of the developing solvent and their vapor, an equilibrium will be established eventually (1). This equilibrium is called chamber saturation. Depending on the vapor pressure of the individual components the composition of the gas phase can differ significantly from that of the developing solvent. While still dry, the stationary phase adsorbs molecules from the gas phase. This process, adsorptive saturation, is also approaching an equilibrium in which the polar components will be withdrawn from the gas phase and loaded onto the surface of the stationary phase (2). Simultaneously the part of the layer which is already wetted with mobile phase interacts with the gas phase. Thereby especially the less polar components of the liquid are released into in the gas phase (3). Unlike (1) this process is not as much governed by vapor pressure as by adsorption forces. During migration, the components of the mobile phase can be separated by the stationary phase under certain conditions, causing the formation of secondary fronts.


• Also called Chamber Saturation

• Low polarity mob. Phase:- no need

• High polar mob. Phase:- desirable

• For reverse phase saturate chamber with polar solvent


LOW SOLVENT

CONSUMPTION

pre-equilibration

with

solvent vapor


Start of

development

CAMAG Twin Trough Chambers offer several ways to improve the results of TLC/HPTLC developing techniques. It allows low solvent consumption, reproducible pre-equilibration with solvent vapor, equilibration performed with any liquid and for any period of time, and development is started only when developing solvent is introduced into the trough with the plate. Twin Trough Chambers are available with stainless steel lid or as a Light-Weight Twin Trough Chamber made from highly transparent sheet glass with a glass lid.


After development, remove the plate and

mobile phase is removed from the plate - to avoid

contamination of lab atmosphere.

Dry in vacuum desiccator - avoid hair

drier because essential oil components may

evaporate.


Detection under UV light is first choice - non destructive.

Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length).

Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF.


CATS STANDARD PROGRAM.

CATS PROGRAM OPTIONS


Non UV absorbing compounds like

ethambutol, dicylomine etc - dipping the plates in

0.1% iodine solution.

When individual component does not

respond to UV - derivatisation required for

detection .


HPTLC 100µm High due to smaller particle

size generated

3 - 5 cm

Shorter migration distance and the analysis time is greatly reduced

Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes

New type that require less amount of mobile phase

Auto sampler

Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type

of densitometer

TLC

250µm

Less

10 - 15 cm

Slower

Silica gel , Alumina

More amount

Manual spotting

Not possible


Pharmaceutical Researches Biomedical Analysis Clinical Analysis Environmental Analysis Food Industry Therapeutic drug monitoring to determine concentration of drug and it’s metabolite in blood, urine etc Analysis of environmental pollutions levels Quantitative determination of prostaglandin’s and thromboxanes in plasma Analysis of nitrosoamines in food and body fluids Determination of sorbic acid in wine Characterization of hazards in industrial waste


(in the name of god) - دانشگاه علوم پزشكي...

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